transEDIT CRISPR/Cas9 lentiviral reagents provide powerful tools for genome editing, offering optimized gRNA designs cloned into a choice of expression vectors and formats for engineering specific gene knockouts. The vectors are designed to select for high gene editing efficiency using direct indicators of Cas9 expression from fluorescent or selection markers as well as efficient delivery as plasmid or lentiviral particles. In addition, complementary markers for co-selection of Cas9 and gRNA ensure efficient expression of both components necessary for Cas9 based genome editing.
transEDIT reagents include lentiviral expression vectors containing specific gRNA targeting your gene of interest in various formats:

The percentage of indel frequency as a measure of targeting efficiency can be determined using the Surveyor assay (Guschin et al., 2010). The mutations introduced by the CRISPR Cas9 reagents result in sequence differences between the chromosomes. PCR amplification of the targeted area amplifies both alleles. Amplicons consisting of one strand from each allele have a bulge where the sequences differ. Treating the PCR product with nuclease digests the amplicon into two fragments. Using a bioanalyzer (or agarose gel) the percent of digested product and percent of cells with indels can be estimated.

gDNA extracted from cells transduced with transEDIT lentiviral vectors (and 1 week of puromycin selection) was amplified with a high-fidelity polymerase and subsequently assayed with Surveyor nuclease. Cas9-mediated cleavage efficiency (indel percent) was calculated based on fraction of cleaved DNA as detected by Bioanalyzer, High Sensitivity DNA Assay. Indel frequency for targeted loci are reported.

High Frequency InDels by Enriching for High Cas9 Expression with Fluorescent Markers

The level of Cas9 expression has been shown to impact the efficiency of indel creation. Duda et al. showed that vectors with fluorescent markers tied to Cas9 expression through a 2A peptide allow the selection of the high Cas9 expressing cells through FACS analysis. Similar selection strategies can be employed with transEDIT Cas9 expression vectors.

Red and Green fluorescent marker expression was tied directly to Cas9 nuclease or nickase expression.
The 2A peptide tag ensures that the Cas9 and marker proteins are expressed at equimolar amounts. Cells were electroporated and cultured for 72 hours. FACS analysis was used to separate the heterogenous populations of cells into groups with low, medium or high fluorescence expression. A, VEGFA was targeted with a vector expressing a single gRNA and Cas9 nuclease linked to GFP via a 2A peptide. The population with the highest fluorescence had approximately 10-fold more cells with an indel mutation detectable by the Surveyor assay. B, two vectors expressing both nickase and a gRNA targeting EMX1 were also tested in combination with a single-stranded donor oligonucleotides (ssODN) that would introduce an EcoRV restriction site. Comparing digestion from EcoRV versus surveyor assay showed that both homologous recombination and nonhomologous end joining were increased significantly in the high expressing population. (Figure adapted from Duda et al, 2014.)